CaCl2 priming promotes sorghum seed germination under salt stress by activating sugar metabolism

Salt stress notably inhibits the germination of sorghum seeds. CaCl2 priming effectively promotes seed germination under salt stress, but the underlying mechanisms remain unclear. This study explored the CaCl2 -primed regulation of sorghum seed germination under salt stress. Hydro-primed seeds (HPS) and CaCl2-primed seeds (CaPS) were cultured under salt stress (150 mM NaCl). The no-primed seeds were cultured in distilled water (no salt) (NPN) or 150 mM NaCl stress (NPS). Primed and unprimed seeds were evaluated for amylase activity, starch content, sugar metabolism, and mitochondrial repair. We found that salt stress significantly inhibited sorghum seed germination and reduced the germination rate. It also decreased amylase activity, starch decomposition, and sugar accumulation during germination, indicating inhibition of sugar metabolism. However, CaCl2 priming reversed the adverse effects of salt stress, increasing amylase activity, starch decomposition, and sugar content. It also up-regulated the expression of genes for phosphofructokinase and other enzymes involved in the glycolytic, tricarboxylic acid cycle (TCA), and pentose phosphate pathways. CaCl2 priming also resulted in the repair and maintenance of mitochondrial structural integrity, contributing to activation of the TCA cycle. In short, CaCl2 priming promotes sorghum seed germination by activating sugar metabolism under salt stress and provides a strategy for improving seed germination during agricultural production.


Introduction
Sorghum (Sorghum bicolor L. Moench) is the fifth most important cereal crop in the world (FAO 2023) and is mainly cultivated on marginal land, which adapts to a poor environment, and has great potential for agricultural development (Karimuna et al. 2020).Sorghum is a subsistence crop that serves as a staple food for people in arid and semiarid regions and is also used as a feedstock for industrial processes such as brewing and bioethanol production (Stagnati et al. 2021).Although sorghum is highly stress-resistant, it is sensitive to salt stress during seed germination, which limits the emergence and establishment of seedlings, thus affecting sorghum production (Zhu et al. 2018).Therefore, promoting the germination of sorghum under salt stress plays a decisive role in improving its cultivation under adverse conditions.
Salinization is a crucial environmental problem restricting crop production (Mbarki et al. 2020).According to statistics, at least 900 million hectares, or approximately 7% of the world's land areas, are affected by salinity (Bijalwan et al. 2021).The high salt concentration increases the osmotic potential of the soil, thus reducing the water absorption by plants.As a result, seed imbibition is inhibited during germination leading to poor seed germination (Gupta and Huang 2014).Several studies in recent years Yifan Xing and Xiaofei Chen have contributed equally to this work.
Communicated by Zhong-Hua Chen.have reported the retardation or inhibition of seed germination under salt stress (Ibrahim 2016;Kubala et al. 2015;Venancio et al. 2020).
Many methods and techniques have been used to improve plant resistance to salt stress.Seed priming is one such technique that plays a positive role in plant stress resistance (Paparella et al. 2015).It involves the treatment of seeds with compounds before germination to induce specific physiological states of seeds.The hydration of seeds triggers the metabolic process activated during early germination, thus accelerating and promoting uniform seed germination (Marthandan et al. 2020).Other seed priming methods include water priming, chemical priming, osmosis priming, and hormone priming.Seed germination is a complex process coordinated and regulated by metabolic, cellular, and molecular activities in the life cycle of higher plants and is a critical period for establishing crop populations (Bourioug et al. 2020).Therefore, seed priming helps plants cope with various stresses by regulating biological processes such as enzyme activities (Gammoudi et al. 2020;Li et al. 2013).
Sugar metabolism is one of the most important physiological processes during seed germination, providing the nutrients and energy (Bewley 1997) required for germination.Seed germination begins with the imbibition of dormant seeds and the mobilization of reserve materials to provide energy for embryo growth, and salt stress affects seed germination by limiting water absorption and inducing ion toxicity, thus affecting the mobilization of reserves in seeds (Lozano-Isla et al. 2018).Starch is the most abundant sugar in cereal seeds, and amylase plays a significant role in decomposing the stored starch.Therefore, amylase activity during seed germination determines the rate of starch decomposition and the supply of energy materials in seeds.Energy and intermediates from sugar metabolism are essential for embryo and seedling growth during seed germination (Cao et al. 2019;Ma et al. 2017).There were found that seeds primed with selenium and salicylic acid enhance glucose metabolism by increasing the activities of pyruvate kinase (PK), hexokinase (HK), phosphofructokinase (PFK), and malate dehydrogenase (MDH) in the tricarboxylic acid cycle (TCA), leading to increased energy generation to promote rice seed germination and seedling establishment under cold stress (Nie et al. 2020).Proteomic analysis of germinating seeds revealed that melatonin priming is critical for maintaining TCA cycle stability and providing more energy, thus improving the germination of aged seeds (Yan et al. 2020).These priming agents act as signaling molecules and critical regulators of sugar metabolism.As a second messenger, Ca 2+ mediates metabolic processes involved in plant growth, development, and stress tolerance (Mulaudzi et al. 2020).Adding 2 mM Ca 2+ to the nutrient solution enhances activities through the glycolytic and TCA cycle and promotes the growth of cucumber roots under hypoxia stress (He et al. 2015).An earlier study showed that enhanced Ca 2+ signaling in the mitochondria up-regulates the pyruvate dehydrogenase (PDH) of the TCA cycle, positively affecting energy production (McCormack et al. 1990).Furthermore, calcium-binding inhibitors reduce PK activity, and PDH complexes (PDC) assist in converting pyruvate to acetyl-CoA, indicating glycolysis and the TCA cycle may be regulated by Ca 2+ in plants (Miernyk et al. 1987).All these studies confirm the essential role of Ca 2+ in regulating sugar metabolism.However, little is known about the calcium-primed regulation of sugar metabolism in sorghum seed germination under salt stress.
It has been confirmed that CaCl 2 significantly promotes the germination of sorghum seeds under salt stress (Hussain et al. 2018).But little is known about the effect of CaCl 2 priming on sugar metabolism during sorghum germination under salt stress.Therefore, this study explored the effects of CaCl 2 priming on starch decomposition, sugar metabolism, and mitochondrial repair during sorghum seed germination under salt stress to reveal the mechanisms underlying CaCl 2 -primed regulation of sorghum germination under salt stress.

Experimental design
The study was conducted in the Sorghum Physiological Laboratory of Shenyang Agricultural University.The sorghum cultivar, Liaoza 15, provided by the Liaoning Academy of Agricultural Sciences, Liaoning Province, China, was used as the experimental material in this study.Seeds of uniform size were selected and disinfected with 5% sodium hypochlorite solution for 5 min.The seeds were washed five times with distilled water and then surface dried.The seeds were primed in distilled water or CaCl 2 solution (1 g/5 mL refers to putting 1 g of seeds into 5 mL of solution) for 12 h in the dark at 25 °C and dried at room temperature (25 °C) to the initial water content (13%, w/w).The preliminary tests showed that the 6 g/L CaCl 2 solution had the best priming effect.The no-primed seeds were cultured in distilled water (no salt) (NPN) or 150 mM NaCl stress (NPS).The hydro-primed seeds (HPS) and CaCl 2 -primed seeds (CaPS) were both cultured under salt stress (150 mM).The seeds germinated in a Petri dish with a double-layer of filter paper.To each petri dish was added 10 mL distilled water or NaCl solution and placed in a completely dark climate incubator at 25 ± 1 °C and 70% humidity.The treatment was repeated thrice with 50 seeds in each treatment.The criterion for seed germination is that the radicle is larger than 2 mm (Zhang et al. 2020).The number of germinating seeds and the germination rate were measured at 6 h, 12 h, 24 h, and 48 h.The shoot length, root length, fresh weight, and dry weight of seedlings were measured for 48 h.Liquid nitrogen was used for rapid freezing, and the germinated seeds were stored at − 80 °C.Amylase activity, starch content, sugar content, and activity of enzymes involved in sugar metabolism were determined.Seeds germinated for 48 h were used for RNA extraction and qRT-PCR.

Determination of germination characteristics, morphological characteristics, and biomass
The root length, shoot length, fresh weight, and dry weight of 10 shots were measured in each treatment.

Determination of starch, total sugar, and amylase activities
Total sugar and starch were extracted with ethanol (80%) and perchloric acid (36 M), respectively, and measured by the anthrone method (Wang and Huang 2015).The extraction and determination method for total amylase has been previously described (Kishorekumar et al. 2007).Amylase was extracted with distilled water, and the reaction mixture (2 mL) was incubated at 30 °C for 5 min.After adding 2 mL chromogenic agent to the sample, absorbance was measured at 540 nm.

Assay for enzyme activities involved in sugar metabolism
Enzyme-linked immunosorbent assay (ELISA) was used to determine the activities of PK, PFK, HK, α-ketoglutarate dehydrogenase, and 6-phosphoglucose dehydrogenase (Peng et al. 2014).More detailed information was as follows.
1 g of pre-cooled sorghum seeds were added into the 5 mL extraction solution.Then they were completely grinded and poured into a centrifugal tube.The sample was centrifuged at 4 °C and 13,400 × g for 10 min.The supernatant was fully reacted with the standard substance at 37 °C for 30 min.After 5 rinses of the ELISA plates with the reaction mixture, 50 μL of reaction mixture was added to the plates again.Then 100 μL of chromogen reagent was mixed at 37 °C for 10 min.Finally, the stop solution was added to the plates, and the measurement of absorbance was at 450 nm in 15 min.The linear regression equation of standard curve was calculated with the concentration of standard substance and its absorbance, and the final enzyme concentration was obtained by substituting sample absorbance into the equation.All above reagents were provided by Changchun Bally Gene Biotechnology Co. Ltd.

Mitochondrial ultrastructure
The mitochondrial ultrastructure was observed in the apical materials of the embryo and radicle, cut into 1 × 1 × 2 mm samples with a sharp blade.The samples were treated as described previously (Nie et al. 2020).Sections were cut with an ultrathin slicer (LEICAEMUC7, Germany), stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (HT7700, Hitachi, Japan).

Extraction of RNA
The germinated seeds were thoroughly and rapidly ground in liquid nitrogen with TRNzol Universal reagent (1 ml for 0.1 g sample).The homogenized samples were left at room temperature for 10 min to separate the nucleic acid-protein complex, followed by centrifugation at 12,000 RPM (about 13,400 × g) at 4 °C for 10 min, and the supernatant was collected for further evaluation.
Chloroform was added to the homogenized sample (0.2 mL for every 1 mL of TRNzol Universal reagent), covered, shaken vigorously for 15 s, and stored at room temperature for 3 min.The sample was centrifuged at 12,000 RPM (~ 13,400 × g) at 4 °C for 15 min, separating the contents into three layers: pink organic phase, middle and upper colorless aqueous phase.The aqueous phase (about 500 μL) contained most of the RNA and was transferred to a new centrifuge tube.Isopropyl alcohol was added to the RNA sample and centrifuged at 12,000 RPM (~ 13,400 × g) for 10 min at 4 °C.The supernatant was collected.Before centrifugation, the RNA precipitate was usually invisible and gelatinous, found mainly on the side and bottom of the tube.The precipitate was washed in 75% ethanol prepared in RNase-free ddH 2 O (using at least 1 mL for every ml of TRNZol universal reagent) by centrifugation at 10,000 RPM (~ 9391 × g) at 4 °C for 5 min.The supernatant was discarded, taking care not to throw out the precipitate.Any remaining solution was centrifuged briefly, and the supernatant was aspirated with a pipette, taking care not to disturb the precipitate.The precipitate was dried at room temperature (taking care not to over-dry it since completely dried RNA is difficult to dissolve, about 2-3 min).Based on the experimental requirement, 30-100 μL RNase-free ddH 2 O was added and mixed well to dissolve the RNA fully (Fu et al. 2019).The gene ID and primer information used for qRT-PCR analysis in this study are shown in Table 1.

Statistical analysis
One-way analysis of variance (ANOVA) was performed using SPSS 18.0 software (SPSS, Inc., Chicago, USA).The least significant difference (LSD) between treatments was assessed using Duncan's new complex range method.Origin 1 3 2018 (Origin Lab, Massachusetts, USA) and Graph Pad Prism 8 (Graph Pad Software, Inc., San Diego, CA, USA) were used for mapping.The data in the chart are expressed as mean ± standard deviation.

Effects of CaCl 2 priming on germination of sorghum seeds under salt stress
The date in Fig. 1 reveal that salt stress significantly inhibited the seed germination rate and suppressed growth of roots and shoots of sorghum seedlings.However, seeds primed with CaCl 2 exhibited sorghum germination under salt stress, significantly improving the growth of roots and shoots.Water priming mitigated damage to germination.The seeds began to germinate after 6 h (Fig. 1A).Compared with NPN treatment, NPS treatment significantly reduced the germination rate at 12 h (78.57%), 24 h (36.67%), and 48 h (21.54%).Compared with NPS treatment, HPS treatment significantly increased the germination rate at 12 h (191.67%) and 24 h (47.37%), while there was no significant difference at 48 h.Compared with NPS treatment, CaPS treatment significantly increased the germination rate at 12 h (325%), 24 h (52.63%), and 48 h (27.45%) (Fig. 1B).NPS treatment significantly increased the average germination time (76.41%) and 50% germination time (92.75%)compared to the NPN treatment.HPS and CaPS treatments significantly decreased the average germination time (32.06% and 37.29%, respectively) and the germination time over 50% (38.73% and 46.29%, respectively) compared with NPS treatment (Fig. 1C, D).Besides, compared to NPN, the germination index of NPS treatment was significantly decreased by 52.32%.Similarly, compared to NPS treatment, the germination index of HPS and CaPS treatments were significantly increased by 64.60% and 99.12%, respectively (Fig. 1E).

Effect of CaCl 2 priming on sorghum seedling growth under salt stress
Salt stress significantly reduced the shoot length (85.59%), root length (67.65%), fresh weight (32.66%), and dry weight (26.65%) of sorghum seedlings (Fig. 2A-D).Compared with NPS treatment, HPS treatment significantly increased the root length (69.69%) and dry seedling weight (27.91%) but had no significant effect on the shoot length and fresh seedling weight.In contrast, the CaPS treatment significantly increased the shoot length (254.99%),root length (90.91%), fresh weight (30.87%), and dry weight (31.7%) of sorghum seeds compared to the NPS treatment, while no significant differences were seen in the root length, fresh seedling weight, and dry seedling weight, between HPS and CaPS treatments, the shoot length was significantly increased (119.98%) with CaPS treatment (Fig. 2A-D).

Effects of CaCl 2 priming on mitochondrial ultrastructure of shoot and root cells
Salt stress destroyed the mitochondrial structure of the shoot and root cells.The mitochondrial membranes of the shoot cells were damaged, and the internal structures disintegrated (Fig. 7B).Similarly, the mitochondrial membranes and crest structure of the root cells were also damaged and disintegrated (Fig. 7F).Compared with NPS, water priming repaired the mitochondrial structure to some extent, with the mitochondrial membranes and crest structures becoming clearer and more complete (Fig. 7C, G).However, CaCl 2 priming provided the best protection to the mitochondrial structures in the shoot and root cells.It countered the damage caused by salt stress, making the mitochondrial membranes and crests visible (Fig. 7D, H).

Discussion
Seed germination is a critical phase in the life cycle of any plant and is also the most sensitive to environmental stress (Weitbrecht et al. 2011).Many studies have reported that salt stress reduces seed germination rate and delays seed germination (Gao et al. 2019;Zeng et al. 2018).In this study, salt stress significantly reduced the number of germinating seeds and the germination index.Moreover, CaCl 2 priming reversed the adverse effects of salt stress, significantly increasing the germination rate and index while decreasing the germination time.Similar study also showed that CaCl 2 priming improves the germination rate of wheat seeds (Islam et al. 2015) and the vigor of quinoa seeds (Hajihashemi et al. 2020) under salt stress.The above studies indicated that CaCl 2 priming promotes the germination of sorghum under salt stress.In addition, the concentration of Cl − in this study did not reach the threshold for functioning (Wu and Li 2019), therefore, Ca 2+ was a key trigger for CaCl 2 priming.The initial stages of seed germination, accompanied by water absorption, involve a series of complex physical and chemical processes, including the activation of various enzyme systems, membrane repair, and degradation of stored nutrients (Bewley et al. 2013).While salt stress reduces seed germination by inhibiting starch hydrolyzing enzymes, pretreatment of seeds counters the adverse effects of salt stress on amylase (Hajihashemi et al. 2020).Besides, seed priming agents, such as aspirin (Hussain et al. 2018), salicylic acid, and hydrogen peroxide (Bouallegue et al. 2017), improve amylase activity and promote starch decomposition and seed germination.In the current study, salt stress reduced amylase activity and inhibited starch decomposition, resulting in seeds with high starch content.The pretreatment of seeds significantly promotes seed germination through increased amylase activity, promoting starch degradation (Li et al. 2013).Salt stress also inhibits sugar formation (Yang et al. 2017).CaCl 2 priming increases the sugar content in seeds, providing the necessary nutrients for germination and seedling establishment (Farooq et al. 2006).In brief, CaCl 2 priming enhanced amylase activity and accelerated starch decomposition as small molecular sugars, providing energy for germination and growth of seeds under salt stress.
Sugar metabolism during seed germination and early seedling growth is critical for seedling vigor, especially under adverse conditions (Hussain et al. 2016).Environmental stress usually leads to significant alterations in sugar metabolism through the altered expression of various genes (Kobylinska et al. 2018).For example, osmotic stress caused by salt can seriously affect the metabolic pathways controlled by enzymes since they are highly dependent on water availability (Awatif and Alaaeldin 2017).Therefore, the reduction of key enzyme activities in sugar metabolism observed in this study may be caused by the decrease in water availability due to saltinduced osmotic stress.Seed priming improves the ability of germinating seeds to regulate osmotic stress and enhance water availability (Ibrahim 2016).This could also explain the enhanced enzyme activity in response to CaCl 2 priming in this experiment.Besides, respiratory metabolism and energy production in the early stages of seed germination depend heavily on the glycolytic pathway (Liu et al. 2018).HXK1, a HK, is a glucose sensor that regulates nutrient and hormonal signaling networks to control the growth and development of Arabidopsis and responds to environmental changes (Moore et al. 2003).The results of the study showed that CaCl 2 priming increased the activities of all glycolytic enzymes, thereby increasing the glycolytic flux, improving salt tolerance, and promoting seed germination.
In addition to enhancing the activities of critical regulatory glycolytic enzymes, CaCl 2 priming also impacts the sugar content.Sugar increases the expression of PK, HK, and PFK in plant tissues (Bouny and Saglio 1996).In the current study, CaCl 2 priming promoted starch metabolism and increased the sugar content in germinating sorghum seeds under salt stress, which might account for the increased PK, HK, and PFK activities.Furthermore, calcium-binding protein inhibitors have been shown to decrease PK activity, while the PDC assists in the conversion of pyruvate to acetyl-CoA.Hence, Ca 2+ potentially regulates the link between glycolysis and the TCA cycle in plants (Miernyk et al. 1987).TCA cycle is the primary component of mitochondrial respiration that connects glycolysis and mitochondrial electron transport chain.It is also a central metabolic process for energy generation (He et al. 2015).A previous study has suggested that CaCl 2 -primed seed germination may be attributed to the activation of the TCA cycle, mitochondrial repair, and biogenesis (Chen et al. 2021).We found that the integrity of the mitochondrial structure was well protected by CaCl 2 priming under salt stress.Mature and dried seeds have poorly differentiated mitochondria, many of which are repaired and biosynthesized during seed imbibition (Bewley 1997).Studies have shown that after seed priming, mitochondrial repair and development can be initiated even when the primed seeds are dried back to their initial water content (Chen and Arora 2013).Sun et al. (2011) reported that the membrane structural integrity of primed seeds is better than that of unprimed seeds, which is consistent with our findings.In addition, the activity of α-ketoglutarate dehydrogenase, a rate-limiting enzyme involved in the TCA cycle, was significantly increased by CaCl 2 priming during seed germination.An increase in mitochondrial Ca 2+ enhances the activities of major ratelimiting enzymes in the TCA cycle, increasing ATP production (McCormack et al. 1990), which may explain how CaCl 2 priming enhances the TCA pathway and promotes seed germination.It is also noteworthy that the pentose phosphate pathway results in the oxidative decomposition of glucose, during which NADPH is produced and used as a reducing agent for biosynthesis (Hou et al. 2006).The activity of 6-phosphoglucose dehydrogenase, a key enzyme of the pentose phosphate pathway, reflects the status of this pathway.In this study, CaCl 2 priming enhanced the expression of the 6-phosphoglucose dehydrogenase gene and its activity during sorghum seed germination under salt stress.The CaCl 2 priming, therefore, reversed the salt stress-induced inhibition of the pentose phosphate pathway, enhanced the glucose metabolism in germinating seeds and promoted germination under salt stress (Fig. 8).
In conclusion, CaCl 2 priming can alleviate the inhibition of sorghum seed germination under salt stress.CaCl 2 priming increased amylase activity leading to higher starch decomposition and increased sugar content during germination, providing the required nutrients for seed germination.Moreover, it increased the expression of genes for key enzymes related to glycolysis, TCA cycle, and pentose phosphate pathway, repairing the mitochondrial structure, thus promoting seed germination under salt stress.

Fig. 1
Fig. 1 Effect of CaCl 2 priming on germination of sorghum seeds under salt stress.A Representative photograph of sorghum.B Germination rate.C Mean germination time.D Time to 50% germination.E germination index.For each treatment, values were obtained from three biological repeats (n = 3).Different letters above the bars are

Fig. 2
Fig. 2 Effect of CaCl 2 priming on sorghum seedling growth under salt stress.A Shoot length.B Root length.C Germinated seed fresh weight.D Germinated seed dry weight.For each treatment, values were obtained from three biological repeats (n = 3).Different letters above the bars are statistically different by a least significant difference test (p < 0.05).'NPN', 'NPS', 'HPS' and 'CaPS' denote noprimed and no stress, no-primed and salt stress, water-primed under salt stress, CaCl 2 -primed under salt stress, respectively

Fig. 3
Fig. 3 Effect of CaCl 2 priming on amylase activity during sorghum germination under salt stress.For each treatment, values were obtained from three biological repeats (n = 3).Different letters above the bars are statistically different by a least significant difference test (p < 0.05).'NPN', 'NPS', 'HPS' and 'CaPS' denote no-primed and no stress, no-primed and salt stress, water-primed under salt stress, CaCl 2 -primed under salt stress, respectively

Fig. 5
Fig. 5 Effects of CaCl 2 priming on glucose metabolism enzyme activities during sorghum germination under salt stress.A Pyruvate kinase activity.B Phosphofructokinase activity.C Hexokinase activity.D α-Ketoglutarate dehydrogenase activity.E 6-Phosphoglucose dehydrogenase activity.For each treatment, values were obtained

Fig. 6
Fig. 6 Effects of CaCl 2 priming on the gene expression of glycometabolism enzymes in sorghum seeds under salt stress.A The expression of PFK2.B The expression of PFK6.C The expression of G6PDH1.D The expression of G6PDH2.E The expression of KGDH1.F The expression of KGDH2.For each treatment, values were obtained from three biological repeats (n = 3).Different letters above the bars are statistically different by a least significant difference test (p < 0.05).'NPN', 'NPS', 'HPS' and 'CaPS' denote noprimed and no stress, no-primed and salt stress, water-primed under salt stress, CaCl 2 -primed under salt stress, respectively

Fig. 7
Fig. 7 Effects of CaCl 2 priming on mitochondrial ultrastructure of sorghum shoot and root cells.Shoot and root cell ultrastructure of NPN (A, E), NPS (B, F), HPS (C, G), CaPS (D, H).Scale bars 500 nm

Table 1
Primers used for qRT-